In this era of growing antibiotic resistance, bacteriophage have garnered much interest for their potential to control pathogenic bacteria, including foodborne pathogens like E. coli O157:H7. However, it is increasingly clear that the interrelationships of phages and their hosts involve many parameters, and the paucity of information regarding phage behavior and infection kinetics under environmental conditions is hindering the implementation of phage therapy and related applications. The specific aims of this proposal include: 1) a systematic examination of the reproduction kinetics of coliphage infecting hosts under metabolic conditions reflecting those found in the bovine intestine;2) the rational development of phage cocktails that can effectively control E. coli O157:H7, through an examination of the key parameters that affect phage reproduction including the interplay between individual phages within a cocktail;3) an examination of the effects of stationary-phase physiology on the infection kinetics of phage being considered for use in O157:H7-targetting cocktails. Our work to date indicates that phage infection of bacteria under anaerobic and/or starvation conditions is markedly different from that observed aerobically in rapidly growing cells. Furthermore, when two different phages co-infect a bacterial cell their interplay can drastically affect phage production by one or both in ways that must be considered in selecting cocktail combinations. The in-depth analysis of key mechanisms controlling coliphage infection in the anaerobic environment of the mammalian gut will also provide an essential foundation for the application of phage therapy in such fields as treating the human diarrheal diseases that have such devastating impacts on both infants and adults in many parts of the world. Through the use of physiological experiments in conjunction with molecular techniques such as the quantification of host and phage gene expression using microarray technology, gel analysis of radioactively-labeled proteins and DNA sequencing, we expect to develop predictive criteria about the inclusion of specific phage within a cocktail and the effects of host physiology on the infection process. Such experiments are very well suited to be carried out by our undergraduates, helping them develop a strong foundation in microbial ecology and physiology, molecular biology, general microbial and anaerobic techniques, and experimental design, along with the satisfaction of contributing new research results of significance to human health. We believe this experience is central to the intellectual development not only of those going on to graduate and professional schools, but also for those who will be involved in government, K-12 teaching, or simply making good personal and societal judgments in the future. PUBLIC HEALTH RELEVANCE: Recent E. coli O157:H7 outbreaks have eroded consumer confidence and again made food safety a major issue in the medical and agricultural arena. In recent years, bacteriophage have garnered substantial interest for their potential to control foodborne pathogens, including O157:H7, but information regarding their infection behavior under non-laboratory environmentally-oriented conditions and in mixed infections with other phages, both explored here, is badly needed. Lessons from this study are also needed for the application of phage therapy in such critical areas as the treatment of the diarrheal diseases that kill so many people world-wide.